Leishmaniasis refers to a group of common parasitic diseases that are highly endemic in many nations including most Middle Eastern countries. Military personnel are at risk for this vector-borne disease. Indeed, leishmaniasis reached epidemic proportions in the US military during the Operations Enduring Freedom (OEF), Iraqi Freedom (OIF) and New Dawn (OND). The ratio of asymptomatic to symptomatic leishmaniasis varies from 6:1 to 98:1 in different studies. It is therefore highly likely that there are many more unrecognized infections in our military personnel than the ~600 reported. Leishmania have the ability to persist in a host even after clinical cure. Indeed, infected humans and other hosts may never clear the infection, even after disease symptoms resolve. Leishmania are known to exert a profound effect on innate and adaptive immune responses of the host during infection. Recent evidence documents that immune effects persist even after cure of disease. Thus, it is highly likely that the immune effects of leishmaniasis extend beyond measurable symptomatic disease. Exosomes are small vesicles released from most living cells that contain protein, RNA and lipids from the cell of origin. Exosomes are now recognized as major vessels transporting immune and other response signals between host cells. Recently it was recognized that exosomes are released from Leishmania parasites. These tiny packages of information may provide clues that explain the profound systemic effects of Leishmania infection. We have already characterized the protein content of exosomes from different life stages of Leishmania infantum, one of the causative agents of leishmaniasis in the Middle East. The current proposal is based on the hypothesis that proteins and small RNAs released in exosomes from Leishmania or from Leishmania-infected cells are responsible for the dramatic and persistent local and systemic effects of leishmaniasis on immune cell responses. These effects could be invoked during active or during asymptomatic infection. Our corollary posits that Leishmania parasites themselves release exosomes whose contents affect the intracellular environment, and which become incorporated themselves into exosomes released from host cells. An understanding of the host- and parasite-derived content of released exosomes, therefore, would provide the basis for novel approaches to diagnosis and therapy of leishmaniasis. Specific aims of this project are: Aim #1. Identify the protein and small RNA content of exosomes, and the pathway of exosome release from macrophages infected with L. major or L. infantum. Hypothesis: Exosomes from leishmania- infected macrophages contain protein and microRNAs that promote non-classical activation of infected macrophages. Aim #2. Identify proteins and small RNAs in the serum and urine exosomes from members of the US military who acquired cutaneous leishmaniasis in Iraq, and determine whether exosome proteins elicit an immune response in humans or in mice infected with L. major or L. infantum. Hypothesis: Exosomes released from Leishmania-infected mammalian cells contain dominant parasite antigens that generate an immune response in the infected individual. Aim #3. Document the immunomodulatory properties of exosomes from parasites, from infected macrophages, or circulating in serum of humans with leishmaniasis. Immune responses will be tested in human macrophages and in mouse models of L. major and L. infantum infection. Hypothesis: Exosomes exhibit functional enzymatic activities that affect the activation state and the microbicidal response of both the infected and bystander host immune cells.